Expression profile and characteristics of a KCNQ5 splice variant.

Shuk Yin Yeung1, Wienke Lange2, Michael Schwake2, Iain Greenwood1
1Ion Channels and Cell Signalling Research Centre, St George’s University of London, London, United Kingdom, 22 Unit for Molecular Cell Biology and Transgenic Research, University of Kiel, Kiel, Germany

Recent work has shown hitherto neuronal Kv7 channels, encoded by the KCNQ gene family, have a significant impact on smooth muscle function in addition to their roles in cardiac and neuronal excitability (Yeung et al, 2007). KCNQ1, 4 and 5 was observed throughout the murine vasculature. Inhibition of these Kv7 channels by a selective blocker XE991 produced contractions in all vessels studied. Furthermore, the Kv7.2-7.5 activator retigabine relaxed vessel precontracted by phenylepherine. The molecular composition of these vascular Kv7 ion channels remains to be fully elucidated, however there is evidence that murine vasculature also expresses a KCNQ5 splice variant similar to that in human brain (Schroeder et al, 2000). This study shows expression of a KCNQ5 splice variant is present in all murine vessels studied, which differs to that of the brain. Furthermore, electrophysiological characterisation of full length Kv7.5 and its splice variant employed XE991 and retigabine.

Female BALB/c mice (6-8 weeks) were sacrificed by cervical dislocation and various blood vessels were removed and immediately stored in RNA Later. The brain was treated in the same manner and used as the positive control. Total RNA was extracted using spin column technology (Qiagen) and reverse transcribed using M-MLV (Yeung et al, 2007). Conventional PCR of thoracic aorta, carotid artery, mesenteric artery and portal vein detected a shorter KCNQ5 gene product than in the brain. Sequence analysis showed the difference was an excision of 27 nucleotides from the vascular preparations, which equated to the whole of exon 9.

Electrophysiological recordings were performed using Xenopus laevis oocytes, which were harvested as in Schwake et al, 2003. They were injected with either full length KCNQ5 (Q5fl) or splice variant (Q5sv) RNA. Current-voltage relationships were recorded in the absence and presence of pharmacological modulators. Under control conditions, V0.5(Q5fl) of activation was approximately –24 mV. A similar value was calculated for V0.5(Q5sv). XE991 (1 – 100 μM) exhibited similar effects on both Q5 currents. 1 μM had no inhibitory effect , whereas 10 and 100 μM reduced the current by approximately 10 and 55% respectively (n=8). At all concentrations of XE991, V0.5 values did not differ significantly from its control. In separate experiments, retigabine (1 – 100 μM) resulted in a similar concentration-dependent increase in current amplitude for both Q5fl and Q5sv channels. Furthermore, 10 and 100 μM retigabine produced a hyperpolarising shift in the V0.5 of activation (5 and 15 mv respectively), that were identical for Q5sv and Q5fl.

These data show a KCNQ5 splice variant present in murine vasculature that forms a functional channel and contributes to vascular reactivity. Its existence serves to further increase the molecular diversity of native K+ channels.

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